in this project, a collaboration with Gregory Goldberg at Washington University St. Louis, we employed single-molecule TIRF to study the motion of single matrix metalloproteinases (MMPs) during the digestion of collagen. MMPs play an important role in physiological collagen processing pathways including tissue remodeling, wound healing and cell migration. However, the mechanistic details of MMP interactions with collagen have been refractory to study due to the complex nature of the collagen substrate and the motion of the MMPs. By tracking individual MMPs on isolated native collagen fibers with high spatial and temporal resolution we could characterize the motion of the MMP on the substrate, and how this motion is coupled to proteolytic activity. This approach has provided detailed mechanistic information for this important class of enzymes. We have, for the first time, observed the complex motion of individual MMPs on collagen fibers and have developed a comprehensive quantitative model describing how this motion is coupled to proteolysis of the collagen fiber. We found that the motion of MMPs on collagen is both biased and hindered diffusion, that there are binding hot-spots for MMPs on collagen periodically spaced 1.3 and 1.5 microns apart, and that the motion of MMPs on collagen is interrupted by two classes of pauses: short exponentially distributed pause of duration 0.4s and long non-exponentially distributed pauses of duration 1 second. Escape from the long pause state is consistent with a kinetic pathway that includes 10 or more kinetic steps each with a forward rate of 10/s. A small fraction (5%) of the long pauses result in the initiation of collagen degradation, which is followed by the rapid and processive degradation of 15 collagen monomers in the fiber. These results were unanticipated and provide unprecedented insight into the interaction of MMPs with collagen while highlighting the unique capabilities of single-molecule methods to measure complex biomolecular processes. The initial measurements and comprehensive modeling are complete and we have submitted the first manuscript. Furthermore, we developed new methodologies to analyze diffusion in single-molecule traces, which are applicable to any single-molecule analysis of diffusion trajectories. Future work on MMP tracking will be focused on improving the temporal and spatial resolution of the tracking in addition to extending the duration of individual trajectories through the use of nitrogen vacancy nano-diamond labels.